DNA cytosine methylation is a keeper of "cellular memory", stable and able to (co-)determine gene expression. De novo cytosine methylation occurs in development and, if activated inappropriately in the adult, can cause disease. Chronic pain often arises without visible insult or persists after tissue injury has healed. In the rodent single nerve ligation (SNL) pain model, L4 and L5 dorsal root ganglion (DRG) neurons and glia become dysfunctional with altered electrophysiology, gene expression, and cellular plasticity. The basic molecular force underlying these alterations is unknown. Here we propose that chronic pain is reflected in - and possibly driven by - DNA cytosine methylation changes in dorsal root ganglion cells. We imply that cytosine methylation may constitute a new conceptual layer to be added to known pain mechanisms. The hypothesis of this proposal is that cytosine methylation in adult DRG neurons and/or glia is plastic;that it is altered by experimental nerve injury;that it (co-)determines gene expression patterns characteristic of the pain state;and that it is a cause of poor patient recovery from chronic pain. Our proposal has two aims. Aim 1: To test if cytosine methylation is globally altered in DRGs of neuropathic pain-rats. The HELP assay (=HpaII tiny fragment Enrichment by Ligation-mediated PCR) will be used to perform a genome-wide analysis of 1 million HpaII tiny fragments (HTF) reflecting the methylation status of ~1.4 million unique HpaII/MspI sites in the L4 and L5 DRGs harvested 14 days after SNL or sham operation. Analyses will identify genomic regions where cytosine methylation is altered and sets of individual loci and/or of large gene regulatory regions where methylation changes are most marked. Aim 2: To test if cytosine methylation is locally altered in predicted regulatory regions. 70 pain genes will be chosen from the literature and from HELP result for in-depth methylation analysis of every CpG around each transcription start site using MassArray, a method of methylation analysis combining base-specific cleavage and mass-spectrometry. Research outcomes of Aims 1 and 2 will be validated in an independent set of animals. If conducted now, the proposed study would be the first to investigate DNA cytosine methylation in pain. PUBLIC HEALTH RELEVANCE: Cytosine is one out of the four building blocks that make up DNA. Unlike the other three building blocks, cytosine can be permanently chemically modified through a process called DNA cytosine methylation. This process leads to "methyl-cytosine", which is often referred to as the "fifth base" of the genome. Methyl cytosine is a keeper of "cellular memory", stable and able to (co-)determine how genes function. De novo cytosine methylation occurs in development and, if activated inappropriately in the adult, can cause diseases like cancer. Chronic pain often arises without a clear cause and can persist after tissue injury has healed. In the rodent model, two small structures of the nervous system located near the spinal cord, the L4 and L5 dorsal root ganglion (DRG) become dysfunctional. The basic force underlying these alterations is unknown. Here we propose that chronic pain is reflected in - and possibly driven by - DNA cytosine methylation changes in dorsal root ganglion cells. We imply that cytosine methylation may constitute a new conceptual layer to be added to our current knowledge about how chronic pain arises. The hypothesis of this proposal is that cytosine methylation in adult DRG neurons and/or glia is plastic;that it is altered by experimental nerve injury;that it (co- )determines gene expression patterns characteristic of the pain state;and that it is a cause of poor patient recovery from chronic pain. Our proposal has two aims. Aim 1: To test if cytosine methylation is globally altered in DRGs of neuropathic pain-rats. We will answer this question using a new technology, which we have developed, the HELP assay. This method combines digestion of DNA, amplification, and binding to 2.1 million probes on a glass slide. It allows us to obtain a picture of cytosine methylation for the entire genome in a single experiment. Aim 2: To test if cytosine methylation is locally altered in predicted regulatory regions. 70 pain genes will be chosen from the literature and from HELP result for in-depth methylation analysis of every CpG around each transcription start site using another method termed MassArray, which has specific advanteages when results with high resolution are desired. Research outcomes of Aims 1 and 2 will be confirmed in an independent set of animals. If conducted now, the proposed study would be the first to investigate DNA cytosine methylation in pain.